MINI-REVIEW

beverage bioflavouring and bioproduction

Antonio G. Cordente1 & Simon Schmidt1 & Gemma Beltran2 & Maria Jesus Torija2 & Christopher D. Curtin3

Received: 3 March 2019 /Revised: 9 April 2019 /Accepted: 9 April 2019

# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Aromatic amino acid metabolism in yeast is an important source of secondary compounds that influence the aroma and
flavour of alcoholic beverages and foods. Examples are the higher alcohol 2-phenylethanol, and its acetate ester, 2-
phenylethyl acetate, which impart desirable floral aromas in wine, beer and baker’s products. Beyond this wellknown
influence on the organoleptic properties of alcoholic beverages and foods, there is a growing interest in understanding and
modulating yeast aromatic amino acid metabolism. The tryptophan derivatives melatonin and serotonin have bioactive
properties and exert positive effects on human health, and aromatic amino acids are also the precursors of products of
industrial interest, such as nutraceuticals, fragrances, and opium-derived drugs. This mini-review presents current knowledge
on the formation of compounds from aromatic amino acids by Saccharomyces cerevisiae, from genetic and environmental
influences on their flavour impacts in alcoholic beverages to their potential as bioactive compounds, and the use of yeast as
microbial factories for the production of com-
mercially relevant aromatic compounds.

Keywords Amino acid . Yeast . Wine . Fermentation . * Antonio G. Cordente

toni.garciacordente@awri.com.au
Aromatic
Introduction
Saccharomyces cerevisiae performs a range of industrial
fermentations that functionally depend upon the ability of
this species to efficiently convert sugars into ethanol and
carbon dioxide. In alcoholic beverages, we prize ethanol,
and in yeast-leavened breads and doughs, carbon dioxide
is most important. While performing this core function, S.
cerevisiae also produces a range of secondary metabolites,
such as esters, volatile fatty acids, higher alcohols, and
volatile sulphur compounds, which contribute significantly
to the flavour and aroma of wine (Cordente et al. 2012),
beer (Holt et al. 2018),
1
The Australian Wine Research Institute, PO Box 197, Glen
Osmond, SA 5064, Australia
2
Facultad de Enología, Universitat Rovira i Virgili, Marcel·lí
Domingo 1, 43007 Tarragona, Spain
3
Department of Food Science and Technology, Oregon State
University, 100 Wiegand Hall, Corvallis, OR 97331-6602, USA
bread (Birchet al. 2013),and a range ofother fermented goods
(Hirst and Richter 2016).
Of these fermentation aroma compounds, higher alcohols
and esters, derived through catabolism of amino acids, are the
most abundant groups (Vilanova et al. 2013). In beverage
fermentations, such as in beer and wine, amino acids
represent a major source of yeast assimilable nitrogen in wort
and grape juice, respectively. These amino acids are taken up
and metabolised by yeast to support growth and generate
biomass. Unsurprisingly then, the concentration and
composition of amino acids can have an important effect on
the aroma complexity of wines (Rapp and Versini 1995) and

beers (Procopio et al. 2015), as well as baker’s products
(Dueñas-Sanchez et al. 2014).
Higher alcohols, also known as fusel alcohols, are
fermentation-derived volatile alcohols with more than two
carbons, and, thus, a higher molecular weight and boiling
point than ethanol. There are three classes of amino acid-
derived higher alcohols: branched-chain, sulphur-containing
and aromatic (Table 1). With the exception of 2-
phenylethanol (2-PE), the aroma contribution of the
individual higher alcohols is not considered to be pleasant,
particularly at high concentrations. On the other hand, at
lower concentrations, these compounds contribute
Table 1 Summary of characteristics of higher alcohols derived from yeast amino acid metabolism in wine
Higher alcohol Amino acid Aroma descriptor
precursor
Appl Microbiol Biotechnol
wide range of commercially relevant metabolites, such as
nutraceuticals, fragrances and opium-derived drugs. We will
establish the current state of knowledge concerning
regulation of S. cerevisiae metabolic pathways, and
environmental parameters affecting formation of higher
alcohols and derivatives, in the context where most
knowledge has been generated—beverage fermentation. We
will also discuss development of S. cerevisiae strains that
increase higher alcohol concentrations, the use of non-S.
cerevisiae species and the potential for synthetic biology to
deliver yeast cell factories for the production of compounds
Other Acetate ester (aroma descriptor)
Aroma Typical
properties
threshold range
(mg/L) (mg/L)

Branched-chain 2-Methyl propyl acetate or

2-Methyl-1-propanol isobutyl acetate (fruity,
(isobutanol) Valine Solvent, chemical 40 25–87 banana)
3-Methyl-1-butanol Leucine Solvent, fusel, marzipan 30 84–333 3-Methyl butyl acetate or
(isoamyl alcohol) isoamyl acetate (banana)
2-Methyl-1-butanol Isoleucine Solvent, fusel, marzipan 30 16–31 2-Methyl butyl acetate (fruity,
(active amyl alcohol) banana)
Aromatic
2-Phenylethanol Phenylalanine Floral, rose 14 10–180 2-Phenylethyl acetate (floral,
(β-Phenylethanol) honey)
2-(4-Hydroxyphenyl)ethanol Tyrosine a Bitter 2-(4-Hydroxyphenyl)ethyl
1–48
(tyrosol) Bees wax, honey-like acetate or tyrosol acetate
2-(Indol-3-yl)ethanol Tryptophan Bitter 2-(Indol-3-yl)ethyl acetate or
(tryptophol) tryptophol acetate
Sulphur-containing Methionine Sweet, potato 1 0.16–2.4
3-Methylthio-1-propanol 3-(Methylthio)propyl acetate or
(methionol) methionol acetate (potato,
sweet)
Adapted from Waterhouse et al. (2016) a Tyrosol has been described as having Bbees wax, honey-like^ aromas in the literature;
however, the original reference could not be found
positively to wine complexity (Rapp and Versini 1995). with industrial applications through metabolic engineering of
Higher alcohols are important precursors for ester production, aromatic amino acid metabolism.
and there is a strong correlation between the formationof Yeast amino acid metabolism: higher alcohols and
acetate estersand the corresponding higher alcohol (Cordente acetate esters
et al. 2012). The acetate esters ofhigher alcohols are important
in enhancing fruity aromas, particularly in young wines Higher alcohols are formed as a by-product of yeast
(Moreno-Arribas and Polo 2009), and even more importantly metabolism, through decarboxylation and reductionfrom
in beer (Holt et al. 2018; Pires et al. 2014) and sake corresponding α-keto acids. These α-keto acids are derived
(Takahashi et al. 2017). from one of two pathways related to amino acid metabolism:
In this mini-review, we will focus on yeast metabolism of catabolism of amino acids present in the fermentation
the aromatic amino acids L-phenylalanine (L-phe), L- substrate via the Ehrlich pathway (Dickinson et al. 2003) or
tyrosine (L-tyr) and L-tryptophan (L-trp),asthere is growing de novo-synthesised amino acids, also known as the anabolic
interest in applications of these products beyond their well- pathway (Nisbet et al. 2014). Therefore, the production of
known influence in the aroma and flavour of alcoholic higher alcohols is linked to both nitrogen (Ehrlich pathway)
beverages and foods. Some of these compounds have putative and carbon metabolism (anabolic pathway) (Fig. 1).
positive effects onhuman health and act asprecursors for a
Appl Microbiol Biotechnol
S. cerevisiae uses ammonium and glutamine as the
preferred nitrogen sources; however, upon their depletion, or
under nitrogen-limiting conditions, yeast can catabolise
assimilated amino acids via the Ehrlich pathway to obtain
essential nitrogen as needed (Hazelwood et al. 2008). The
first step in the Ehrlich pathway involves the transamination
of the amino acid to form the corresponding α-keto acid
analogue, and the aromatic aminotransferases Aro8p and
Aro9p are specialised for L-trp, L-phe and L-tyr
transamination (Iraqui et al. 1998).
InthesecondreactionoftheEhrlichpathway,α-ketoacidsare
decarboxylated to yield the corresponding aldehydes. The
In the final step of the Ehrlich pathway, each fusel
aldehyde is either reduced into the corresponding higher
alcohol by an alcohol dehydrogenase or oxidised by an
aldehyde dehydrogenase to form the fusel acid (Fig. 1). The
ratio of fusel acids to fusel alcohols is dependent on the
redox status of the cell, with anaerobic fermentative
conditions, like those found in wine and beer fermentation,
favouring formation of higher alcohols (Vuralhan et al.
2003). Under these conditions, the formation
ofhigheralcohols through the Ehrlich pathwaymay provide
an alternative mechanism for the oxidation of excess NADH
formed during fermentation, to that of glycerol formation by
glycerol-3-phosphate dehydrogenase (Hazelwood et al.
2008; Vidal et al. 2015), thus helping to maintain the redox
balance. Alternatively, the formation of higher alcohols may
represent a mechanism for detoxification of aldehydes
produced by the Ehrlich pathway (Boulton et al. 1995).
Recent studies have also shown that aromatic higher
alcohols, specifically 2-PE, may have important roles as
signalling molecules: as attractants for insect vectors to
decarboxylation of 2-phenylpyruvate to form 2-
phenylacetaldehyde is mainly catalysed by the
phenylpyruvate decarboxylase Aro10p (Vuralhan et al.
2003).
Fig. 1 The Ehrlich pathway in yeast. This catabolic pathway (shaded)
consists of three steps that lead to the formation of higher alcohols from
assimilated amino acids. Once formed, the higher alcohols can be
esterified to form the corresponding acetate esters. The α-keto acid could
also be derived during the biosynthesis of amino acids from a carbon
source, known as the anabolic pathway. Enzymes involved in the
different reactions are indicated in italics; AadX refers to aryl alcohol
dehydrogenases (Aad3, 4, 6, 10, 14–16)
fermenting fruits (Dzialo et al. 2017) and in the quorum-
sensing response of yeast, a process of cell-to-cell
communication used by populations to assess their density
and change growth behaviour accordingly (Avbelj et al.
2015).
Once formed, higher alcohols can be converted into their
corresponding acetate esters in a condensation reaction
between the higher alcohol and acetyl-CoA (Fig. 1),
mediated by the alcohol acetyltransferases encoded by ATF1
and ATF2 (Mason and Dufour 2000). Even though acetate
esters are present at lower concentrations than their
corresponding higher alcohols, they have a significantly
higher impact in beverage aroma due totheir lower sensory
thresholdsand their positive fruity and floral-associated
aromas (Table 1).

Influence of metabolites derived from
aromatic amino acids in organoleptic
properties of alcoholic beverages
The aroma of 2-PE is usually described as floral or rose, and
it is considered a positive attribute in wine, beer and cider
(Hirst and Richter 2016) (Table 1). Several studies have
shown that 2-PE can be an important contributor to the
aroma of different white and red wine varieties, such as
Pinot Noir (Fang and Qian 2005), Merlot and Cabernet
Sauvignon (Gurbuz et al. 2006) or Gewürztraminer (Guth
1997a). Concentrations of 2PE in wine are usually above its
odour activity threshold, while concentrations of 2-PEA
(rose and honey aroma) are usually around or below its
odour threshold (Guth 1997b; Vilanova et al. 2013). Besides
2-PE, the other major aromatic higheralcohol inwineis
tyrosol (Bertelli etal. 2002; Griset al. 2011), which
contributes substantially to the total phenolic content of
white and sparkling wines (Chamkha et al. 2003; Peña-Neira
et al. 2000). Tryptophol is the most abundant indolic
compound produced by yeast during alcoholic fermentation,
and concentrations in wine are usually below 10 mg/L
(Monagas et al. 2007).
There is growing evidence that tyrosol and tryptophol
might influence the mouthfeel properties of fermented
beverages, as both compounds have been associated with
bitterness in wine, sake and beer (Gawel et al. 2018; Soejima
et al. 2012; Szlavko 1973). In wine, it has been reported that
tyrosol could impart bitterness even at typical
concentrations of 20–30 mg/ L, particularly in sparkling
wines (Jackson 2009; SáenzNavajas et al. 2012).
Despite the potential for tyrosol and tryptophol to induce
a bitter finish, Szlavko (1973) found that ale beer drinkers
preferred lager beers containing several times the original
level of tryptophol, mimicking the typical concentrations
found in ale beers. Similarly, sake brewed with a tyrosol-
overproducing strain was found to have positive
organoleptic effects due to its Btaste-sharpening^ effect
(Soejima et al. 2012). Acetate esters, derived from tyrosol
and tryptophol, have been identified in wine and sake
(Güntert et al. 1986; Izquierdo Cañas et al. 2008), and
tryptophol acetate has been positively correlated with a
pleasant bitter taste in sake (Takahashi et al. 2016).
Recently, it has beenreportedthat the sulfonation
ofindolecontaining metabolites, such as tryptophol or indole
lactic acid, which occur in wine with ageing, might have an
important effect on the quality of white aged wines
(Arapitsas et al. 2018). Indole-3-acetic acid is considered a
potential precursor of 2-aminoacetophenone, responsible for
Buntypical ageing off-flavour^ in white wines (Schneider
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2014). Another tryptophan derivative, indole, has been
linked to Bplastic/chemical-like^ off-flavours (Arevalo-
Villena et al. 2010), and formation above its odour threshold
in wine has been associated with Bstuck^ or Bsluggish^
fermentations. Even though S. cerevisiae has the ability to
produce small quantities of indole during fermentation, it is
not clear whether its accumulation in wine is derived
directly from yeast metabolism itself or from non-
Saccharomyces species or bacteria that might grow under
these circumstances.
Bioactive compounds derived from yeast
metabolism of aromatic amino acids
In wines, research on bioactive compounds has largely been
focused on polyphenols, mainly flavonoids (such as
anthocyanins and flavan-3-ols) and non-flavonoids (such as
resveratrol, cinnamates and gallic acid), due to their
antioxidant capacity. However, some recent studies have
focused on metabolites derived from aromatic amino acids
that are putative bioactive molecules, including tryptophol
and tyrosol, and the indolic compounds melatonin and
serotonin (FernándezMar et al. 2012; Mas et al. 2014).
Tyrosol and its derivative hydroxytyrosol are found
abundantly in extra virgin olive oil. These compounds have
been linked with antioxidant and anti-inflammatory
properties (Bertelli et al. 2002; Gris et al. 2011) and
antimicrobial properties against pathogenic bacteria (Cueva
et al. 2012) and Candida species (Monteiro et al. 2017), as
well as cardioprotective properties (Marhuenda et al. 2017;
Samuel et al. 2008; Thirunavukkarasu et al. 2008).
Moreover, hydroxytyrosol has shown positive results
against not only cardiovascular diseases but also cancer and
acquired immunodeficiency syndrome (Vilaplana-Perez et
al. 2014). The presence of hydroxytyrosol in wines has been
related not only to yeast activity during alcoholic
fermentation (AlvarezFernandez et al. 2018) but also to the
hydroxylation of tyrosol to hydroxytyrosol by the enzyme
polyphenol oxidase present in grapes (Garcia-Garcia et al.
2013).
Tryptophol has a sleep-inducing property, as observed in
mice (Cornford et al. 1981). This may be due to subsequent
formation of serotonin and melatonin, for which tryptophol
is a precursor, or it may serve as a functional analogue of
these compounds (Mas et al. 2014). Moreover, tryptophol
has been shown to inhibit amyloid fibrillation, associated
with a number of neuronal pathologies (Morshedi et al.
2007). Like tyrosol, tryptophol displays antimicrobial
properties against pathogenic bacteria (Cueva et al. 2012).
Appl Microbiol Biotechnol
The indolic compounds melatonin and serotonin, on the
other hand, have attracted the most interest due to their
bioactivity in humans. In addition to its primary function as
neurohormone that regulates circadian rhythms, melatonin
also has a putative protective effect against
neurodegenerative diseases, such as Alzheimer’s and
Parkinson’s diseases and angiogenesis (reviewed by
Hornedo-Ortega et al. 2016, and shows a powerful
antioxidant activity (Reiter et al. 2016). However, its role in
yeast still needs to be elucidated, and recent studies have
reported that melatonin can act as an antioxidant compound
in yeast (Vazquez et al. 2018). Serotonin is a
neurotransmitter and a precursor of melatonin (Hornedo-
Ortega et al. 2016), also involved not only in various
physiological functions but also in pathological states
(Apetrei 2016). Both melatonin (Kocadagli et al. 2014;
Rodriguez-Naranjo et al. 2011) and serotonin (Wang et al.
2014) have been recently detected in wine and beer.
Melatonin is present in grapes (Iriti et al. 2006), but it is also
produced by yeast involved in the fermentation process
(Fernandez-Cruz et al. 2017; Rodriguez-Naranjo et al.
2012). While the role of yeast and bacteria in the formation
of serotonin has not yet been elucidated, it was detected at
mg/L quantities post malolactic fermentation (Wang et al.
2014), particularly when Lactobacillus plantarum was used
as inoculum (Manfroi et al. 2009). Although the occurrence
of these compounds in fermented beverages is low,
especially in the case of melatonin (ng pg/mL), these
concentrations have been described as contributing
sufficiently to the dietetic intake to exert measurable effects
(HornedoOrtega et al. 2016).
Physicochemical conditions affecting the
formation of higher alcohols
Nitrogen and lipid composition of grape must, along with
fermentation temperature, are some of the factors that
contribute considerably to variations in the profile and
concentrations of aromatic compounds (Bordiga et al. 2016;
Giudici et al. 1990; Rollero et al. 2015).
The production of aromatic alcohols is highly affected by
nitrogen concentration and composition of grape musts.
Nitrogen starvation increases the production of higher
alcohols (Beltran et al. 2005; Carrau et al. 2008), probably
due to the higher levels of α-keto acids produced under these
conditions, which cannot betransaminated and are in
turnconverted into higher alcohols (Hazelwood et al. 2008).
High concentrations of ammonium, as well as the presence
of other preferred nitrogen sources, repress the expression
of transaminases, decarboxylases and dehydrogenases
involved in each of the steps required for the production of
aromatic alcohols (Carrau et al. 2008; Godard et al. 2007),
resulting in lower levels of those alcohols. On the other
hand, ARO9, ARO10 and their transcriptional activator
ARO80 are induced by the presence of L-trp, L-phe or L-tyr
(Godard et al. 2007; Iraqui et al. 1998). Thus, increased
concentrations of the specific precursor amino acid result in
greater formation of the corresponding higher aromatic
alcohol through the Ehrlich pathway (Bordiga et al. 2016;
Hernandez-Orte et al. 2002).
The aromatic higher alcohols tryptophol, 2-PE and
tyrosol regulate morphogenesis during nitrogen starvation
and act as quorum-sensing molecules in S. cerevisiae
(Avbelj et al. 2015; Wuster and Babu 2010). The production
of 2-PE and tryptophol is regulated by cell density, and
when the growing population reaches a specific density or
quorum, the expression of ARO9 and ARO10 is up-regulated,
stimulating the production of aromatic higher alcohols
(Chen and Fink 2006). In addition, tryptophol has an auto-
stimulation behaviour, creating a positive feedback loop by
activating the transcription factor Aro80p and consequently
ARO9 and ARO10 expression (Chen and Fink 2006) (Fig. 2).
Recently, González et al. (2018) reported that low glucose
might inhibit the formation of higher alcohols, which may
be explained by lower yeast cell density in those conditions.
The influence of lipid composition, and mainly
phytosterols, in the synthesis of aromatic compounds has
been recently reported (Rollero et al. 2016), revealing that
an increase in availability of phytosterols favours the
formation of higher alcohols but causes a decrease in the
production of esters. Indeed, Beltran et al. (2008) observed
that the production of isoamylic alcohols and 2-PE was
much higher in natural grape must fermentations than in
synthetic medium fermentations.
Fermentation temperature is another variable that plays
an important role in the final concentration of fermentative
aromas in wine or beer, affecting the metabolic activity of
yeast as well as the expression of genes implicated in aroma
compound synthesis (Beltran et al. 2006; Deed et al. 2017;
Molina et al. 2007). Although in wine fermentation, low
temperature seems to increase the concentration of esters
(Beltran et al. 2006; Molina et al. 2007), in beer production,
the levels of these compounds increase with temperature
(Verstrepen et al. 2003). Indeed, different esters and yeast
strains may show different temperature dependencies,
resulting from the rather complex regulation of ATF1 (Holt
et al. 2018). On the other hand, there is no consensus on the
effect of temperature on the synthesis of higher alcohols.
Molina et al. (2007) showed that in wine fermentations only
the concentration of 2-PE increases with increasing

temperature, whereas Beltran et al. (2008) observed an
increase in the concentration of all higher alcohols.
Genetic determinants affecting aroma
formation from aromatic amino acids and
breeding strategies
Formation of higher alcohols and acetate esters derived from
aromatic precursors has been found to vary substantially
amongst S. cerevisiae strains (Deed et al. 2017; Giudici et al.
1990).Understanding the genetic determinants
involvedinthe formation of aromatic compounds is essential
for the development of industrial yeast strains capable of
generating enhanced aroma profiles. In recent years, the use
of quantitative trait loci (QTL) mapping strategies has
allowed the identification of alleles involved in the
formation of these aromatic compounds. Steyer et al. (2012)
identified allelic variations in ABZ1 affecting the production
of 2-PE and 2-PEA during fermentation. Abz1p is involved
in the synthesis of paminobenzoic acid from chorismate, the
last common intermediate of aromatic amino acid
biosynthesis (Fig. 2). Recently, two natural alleles that
modulate the formation of both 2-PEA and 2-PE in
industrial strains were identified: a dominant allele of FAS2,
which encodes the alpha subunit of the fatty acid synthetase
Appl Microbiol Biotechnol
complex; and a truncated allele of TOR1, the protein kinase
subunit of TORC1, a complex that regulates cell growth in
response to nutrient availability (Trindade de Carvalho et al.
2017). FAS2 had previously been implicated in modulating
aroma compound formation, since another dominant
mutation in this gene results in the overproduction of ethyl
hexanoate (apple-like flavour) (Akada et al. 2001).
Interestingly, allelic differences in one of the targets of
TORC1, GLN3, might also explain differences in 2-PE
formation between yeast strains (Chen et al. 2017).
Recently, a total of 55 QTLs in the yeast genome that
influence the formation of 30 volatile secondary
metabolites, including 2-PE, were identified (Eder et al.
2018).
Extensive research has also been carried out to breed
industrial yeast strains with a deregulated amino acid
Fig. 2 Pathways associated with the metabolism of aromatic amino
acids. Endogenous aromatic amino acid anabolic and catabolic
pathways are indicated with full black arrows; enzymes and
metabolites naturally produced by yeast are shown in black font.
Overexpression of heterologous enzymes (blue arrows) or pathways
(thick blue arrows) and metabolites derived from these pathways are
indicated in blue font. Dotted red lines indicate the major allosteric
checkpoints in the pathway: inhibition of Aro3p by L-phe and Aro4p
and Aro7p by L-tyr. Overexpression of modified Aro4pK229L and
Aro7pG141S results in tyrosine-insensitive enzymes. In addition, Aro7p
is activated by L-trp (green dotted lines). The biosynthesis of aromatic
amino acids starts with the condensation of two glucose-derived
Appl Microbiol Biotechnol
metabolites erythrose-4phosphate (E4P) and phosphoenolpyruvate
(PEP) and followed by a series of enzymatic reactions which lead to
the formation of chorismate (shikimate pathway). Then, chorismate
can enter the L-trp biosynthesis branch or converted by Aro7p into
prephenate, the last common precursor for L-tyr/phe biosynthesis.
Tryptophol, tryptophan and cell density upregulate the expression of
ARO9 and ARO10, which is dependent on the transcription factor
Aro80p (green lines). The formation of p-coumaric acid follows a 1-
metabolism to enhance the flux of carbon towards the
aromatic amino acid pathway (Cordente et al. 2018; Koseki
et al. 2004; Oba et al. 2005). Key enzymes of the aromatic
biosynthetic pathway are subject to feedback inhibition by
terminal amino acidproducts (Braus1991) (Fig. 2),and the
use of toxic amino acid analogues allows the selection of
cells with decreased feedback inhibition of amino acid
synthesis, thereby causing amino acid overproduction and
metabolic overflow to corresponding higher alcohols. In
particular, increased production of isoamyl alcohol, tyrosol
and 2-PE has been achieved by selecting yeast mutants with
resistance to toxic fluorinated amino acid analogues of L-
leucine (Oba et al. 2005), L-tyr (Koseki et al. 2004) and L-
phe (Akita et al. 1990; Cordente et al. 2018; Dueñas-
Sanchez et al. 2014), respectively. Two fluorinated
diastereomers of phe have been widely used, p-fluoro-DL-
phenylalanine (PFP) and o-fluoroDL-phenylalanine (OFP),
which selectively target two different enzymatic activities of
the pathway. PFP-resistant mutants usually present
mutations in TYR1, accumulate both 2-PE and tryptophol,
and produce less L-tyr (Akita et al. 1990; Cordente et al.
2018; Fukuda et al. 1991a). OFP-resistant strains also
accumulate 2-PE, and these usually present mutations in
ARO4, which releases the enzyme from feedback inhibition
by L-tyr (Cordente et al. 2018; Fukuda et al. 1991b). Strains
obtained by these non-GM methods have the potential to be
used by the beverage and food industry.
Even though the use of modern breeding technologies
involving genetic engineering of the host organism, such as
selfcloning, or more recently CRISPR/Cas9, has the
potential to revolutionise the generation of industrial strains
with improved characteristics, currently, there are social,
ethical and legislative barriers preventing the use of these
techniques in the food and beverage sectors. Even though in
countries like Japan, self-cloned yeast strains are not
considered genetically modified organisms, and several
examples of these strains can be found in the literature
(Ikeda et al. 2018; Kitagaki and Kitamoto 2013), these
strains are rarely used in industrial sake brewing (Negoro et
al. 2016). It is uncertain whether in the futurethe use
ofgenetic engineeredstrains inthe beverageand food sectors
would be more acceptable by the wider public. In the
meantime, the development of industrial yeast strains with
improved characteristics is restricted to more traditional
step conversion from L-tyr or a 2-step conversion from L-phe. p-
coumaric acid is the precursor of a series of biotechnological important
compounds (resveratrol, anthocyanins and raspberry ketone). L-tyr and
4-hydroxyphenylacetaldehyde (4-HPAA) are precursors of
benzylisoquinoline alkaloids, while dehydroshikimate (DHS) is the
precursor of vanillin and muconic acid. DAHP, 3-deoxy-D-
arabinoheptulosonate-7-phosphate; 4HPP, 4-hydroxyphenylpyruvate
breeding methods, such as hybridization or mutagenesis and
selection, and more recently to the use of selected cultures
of non-Saccharomyces yeast or adaptive evolution
techniques.
Yeast as cell factories for the production of
aromatic compounds
The aromatic amino acid biosynthetic pathway is a source
of many commercially relevant chemicals, with diverse
industrial applications, as reviewed by Suastegui and Shao
(2016). Some ofthese chemicals include flavour compounds
(vanillin, raspberry ketone and 2-PE), pain-management
pharmaceuticals (benzylisoquinoline alkaloids) or
nutraceuticals (resveratrol), and S. cerevisiae has been
widely used as a microbial factory for the production of
these compounds. Metabolic engineering of yeast generally
involves the enhancement of the flux of carbon into the
aromatic amino acid biosynthetic pathway, together with the
introduction of the heterologous biosynthetic pathway
needed for the production of the specific compound being
targeted.
The most common strategies to optimise flux into the
aromatic amino acid pathway include the following: (a)
deregulation of key allosteric checkpoints in amino acid
biosynthesis by overexpressing mutant versions of ARO4
and/or ARO7, which are insensitive to L-tyr levels; (b)
removing competing pathways by deleting key enzymes of
the Ehrlich pathway; (c) overexpressing mutant versions of
ARO1 to stop the conversion of intermediates in the
shikimate pathway; and (d) increasing the availability of the
main carbon precursor of the aromatic pathway, erythrose-
4-phosphate (Fig. 2).
The L-phe/L-tyr pathway has been modified for the de
novo production of the benzylisoquinoline alkaloids
thebaine and hydrocodone (Galanie et al. 2015), the
antioxidant resveratrol (Li et al. 2015), the plant pigment
anthocyanins (Levisson et al. 2018), and the flavouring
agent raspberry ketone (Lee et al. 2016). Salidroside, a
plant-derived glucoside of tyrosol with diverse biological
properties, has been synthesised to high titers in yeast by
optimising the endogenous L-tyr biosynthetic pathway and
heterologous expression of plant glucosyltransferases (Jiang

et al. 2018). The rose-like aroma compound 2-PE is an
important fragrance ingredient in the food, cosmetic and
perfume industries, as well as a preservative and a
disinfectant—its market is expected to reach US$700
million by 2019 (Pandal 2014). Yeast bioconversion of a
fermentation media with L-phe as the sole nitrogen source
has proved a promising alternative to the synthesis of 2-PE
using chemical methods. This, in combination with different
in situ extraction methods, to counter the toxicity of
2PEtoyeast,hasyieldedtitersof16g/L(Okuniewskaetal.2017).
By engineering the L-trp branch in yeast using
heterologous enzymes, it was also possible to produce high
levels of the mammalian hormone melatonin (Germann et
al. 2016), which is naturally produced by S. cerevisiae in
μg/L concentrations.
Harnessing other yeast species for aroma
production
Although the pathways leading to the formation of flavour
compounds have been mainly studied in S. cerevisiae,
several studies indicate that the Ehrlich pathway is
conserved across a range of yeast genera and species
(Gamero et al. 2016; González et al. 2018; Stribny et al.
2015).
Global volatile compound analyses performed on other
Saccharomyces species with interesting oenological
properties revealed that Saccharomyces kudriavzevii
produced relatively high concentrations of higher alcohols,
whereas Saccharomyces uvarum excelled in the production
of acetate esters (Stribny et al. 2015). Differences found
amongst the Saccharomyces species during aroma formation
may be due, to some extent, to different enzymatic activities
and properties of the alcohol acetyltransferases Atf1p and
Atf2p and the decarboxylase Aro10p (Stribny et al. 2016a,
b). These species typically struggle to ferment to completion
in high-sugar starting materials, such as grape must, but their
potential impacts can be harnessed through interspecies
hybridisation with robust S. cerevisiae strains. For example,
newly generated interspecific wine yeast hybrids introduce
flavour and aroma diversity to wines, particularly
Saccharomyces mikatae/S. cerevisiae hybrids that produce
elevated concentrations of both 2-PE and 2-PEA (Bellon et
al. 2013).
In addition, several non-Saccharomyces wine yeast
species are of increasing oenological interest, such as
Hanseniaspora uvarum, Hanseniaspora vineae,
Metschnikowia pulcherrima, Zygosaccharomyces bailii,
Hanseniaspora guilliermondii, or Torulaspora delbrueckii.
Appl Microbiol Biotechnol
Although, in general, these nonSaccharomyces species
synthesise lower amounts of aromatic higher alcohols than
S. cerevisiae, when used in coinoculation with S. cerevisiae,
they result in wines with comparable and even higher
amounts of 2-PE than those obtained with S. cerevisiae alone
(Azzolini et al. 2015; Barbosa et al. 2015). Co-inoculation
of non-Saccharomyces yeasts alongside S. cerevisiae is also
an emerging trend to enhance beer flavour, for example T.
delbrueckii was shown to increase formation of several
higher alcohols and some acetate esters (Canonico et al.
2017).
The potential of non-conventional yeast to produce
aromatic compounds was systematically analysed amongst
a collection of 143 different species (Gamero et al. 2016).
The diversity of aroma profiles was significant, and,
interestingly, some of the strains were able to produce higher
concentrations of aroma compounds than reference
Saccharomyces strains, such as strains of the species
Starmera caribaea (isolated from cacti) or H. guilliermondii
(isolated from grapes). Notably, those species, together with
H. vineae, produced significantly more 2PEA than S.
cerevisiae (Gamero et al. 2016; Lleixa et al. 2016). These
results suggest that the pathways involved in the formation
of these flavour compounds, such as the Ehrlich pathway, or
the specific enzymes responsible for ester synthesis, are also
present in non-conventional yeasts. However, the regulation
of their expression, the functionality of the enzymes and
even the number of gene copies in the genome are different
in nonconventional yeasts compared to Saccharomyces
species (Giorello et al. 2019; Seixas et al. 2019).
Future directions
While much progress has been made in understanding yeast
metabolism of amino acids, much work remains. Until
recently, the main interest in amino acid metabolism was
driven by the importance of the amino acid-derived higher
alcohols and acetate esters, in the aroma and flavour of
alcoholic beverages and foods. One of the limitations when
studying flavour compound production is that it represents a
non-selectable and polygenic trait, which has limited studies
concerning the genetic mechanisms responsible for the
variations observed between yeast strains in the production
of these compounds. It is not surprising that until recently
the breeding of S. cerevisiae strains with altered formation
of aromatic compounds relied in techniques that do not
require a previous knowledge of the genetic determinants
responsible for their formation, such as the use of toxic
Appl Microbiol Biotechnol
analogues of amino acids to isolate strains with altered
amino acid metabolism.
The development of low-cost and powerful sequencing
technologies has allowed the use of QTL mapping strategies
to identify novel alleles involved in the formation of
aromatic compounds by yeast, such as TOR1, FAS2 or ABZ1.
Typically performed by genetic crossing between two parent
strains, a limitation of QTL can be its specificity to the
strains involved. Treusch et al. (2015) utilised a round-robin
approach with 12 strains to study signalling pathway-related
phenotypes in S. cerevisiae, enabling the identification of
important variants across the genetic and phenotypic
diversity of the species. Similarly, genome-wide association
applied to a large collection of sequenced and phenotyped
strains revealed that copy-number variants were an
important driver of certain traits (Peter et al. 2018).
Approaches with such breadth would likely provide great
insight into aromatic compound production; however, a
major bottleneck remains the ability to measure the
metabolite of interest for such large populations. Some of
the compounds derived from aromatic
aminoacids,suchasmelatonin,serotoninorindole,areproduce
d in low concentrations and require expensive and complex
analytical techniques for their quantification; and there is a
limitation in the ability to use high throughput methods for
their measurement, which might be necessary when
screening progeny from QTL and/or yeast libraries.
Due to the growing interest in S. cerevisiae as a microbial
factory for the production of aromatic compounds with
industrial applications, new and more powerful methods are
being developed to quantify these compounds reliably.
Recently, a fast HPLC-UV method has been developed to
measure more than 20 metabolites of the shikimate/Ehrlich
pathways at low levels (Lai et al. 2017). In addition,
melatonin-responsive biosensor strains have been
developed, which could be expanded to detect other related
compounds (Shaw et al. 2018). Finally, laserenabled
ambient ionisation mass spectrometry techniques could be
used for rapid yeast library evaluation of aromatic
metabolites directly from colonies on agar plates, and
without any extraction or sample preparation (Fang and
Dorrestein 2014).
Beyond the well-studied products of the Ehrlich pathway,
in yeast and within the fermented beverage context, there is
a lack of research concerning aromatic amino acid
derivatives. While there is a growing interest in increasing
the levels of compounds with positive bioactive properties,
such as serotonin and melatonin, other aromatic-derived
compounds have negative impact in the quality of fermented
beverages if present above their odour detection thresholds.
Little research has been performed in studying the
conditions leading to the formation of the off-flavours
indoleand2-
aminoacetophenoneinwine.Similarly,thepotential role in
bitterness of tyrosol, and of tryptophol and its sulfonated
derivative, has not been studied in detail.
Even for the Ehrlich pathway, little is known about the
formation of the higher alcohol methionol from the
sulphurcontaining amino acid methionine. Many similarities
exist between the catabolism of methionine and those of the
aromatic amino acids, and Aro8p seems to be crucial in
modulating the formation of both methionol and the
undesirable sulphur compound methanethiol (Deed et al.
2019). Therefore, it might be possible to modulate the
formation of some of these volatile sulphur compounds by
further studying aromatic amino acid metabolism.
In conclusion, from just a few amino acids, S. cerevisiae
can derive a range of aromatic products that enhance
fermented beverage flavour, and others that have various
industrial or health applications. Further research into the
environmental and genetic factors that determine levels of
their production and research that harnesses bioconversion
potential of non-Saccharomyces yeasts, whether in their
own right or as DNA donors for engineered strains, will
enhance our ability to make flavourful beverages and
develop new industrial processes.
Acknowledgements The Australian Wine Research Institute (AWRI),
a member of the Wine Innovation Cluster in Adelaide, is supported by
Australia’s grapegrowers and winemakers through their investment
body Wine Australia, with matching funds from the Australian
Government. GB and MJT thank the Ministry of Economy, Industry
and
Competitiveness, Spain (Project AGL2016-77505-C3-3-R), for
financial support. CC is supported by funds from the New Zealand
Winegrowers, the Oregon Wine Board and the Agricultural Research
Foundation.
Compliance with ethical standards
Competing interests The authors declare that they have no conflicts of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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